The invention relates to the area of materials research or analysis using electrical means and relates in particular to a device for selective preconcentration of analytes contained in an electrolyte and also to a method for selective preconcentration of analytes contained in an electrolyte.
There are mainly two known methods for separating analytes contained in an electrolyte with the aid of microfluidic systems.
The first method is capillary electrophoresis. It is generally performed by a microfluidic network into which an electrolyte and a sample containing analytes are injected. This network may include a number of reservoirs connected to at least one long microchannel and/or to a network of microchannels having specially arranged intersections to allow a certain quantity of analytes to be injected into the center microchannel. The application of an electric field to this same channel, known as a separation channel, after the injection phase is responsible for the migration of the analytes. Under an electric field, the charged particles move in a liquid medium at a velocity defined by the field and by the mass and charge of the particles (electrophoresis). The velocity of the particles in the liquid is proportional to the electric field, the proportionality constant being called “electrophoretic mobility.” At the solid/liquid interface, a double ion layer formed of a fixed ion layer (surface charge) and a mobile ion layer (diffuse layer in the liquid) forms spontaneously. Under an electric field, the ions in the mobile layer migrate, bringing about general movement of the liquid by viscosity (electro-osmosis). This movement takes place in a single block and its velocity is also proportional to the electric field. The proportionality constant between the velocity of the fluid and the electric field is called electro-osmotic mobility. The concomitant action of electrophoretic migration (velocity of ions in liquid) and the electro-osmotic liquid flow (velocity of liquid) generated by the potential difference acts on the ions contained in the fluid, ensuring that they are carried through the separation channel. The total velocity of an ion in a microchannel subjected to an electric field is hence proportional to the electric field. The proportionality constant is the total mobility of the ion which is the sum of its electrophoretic mobility (unique to each ion) and the electro-osmotic mobility (identical for all ions and dependent on the characteristics of the solid-liquid interface).
Detection of the various analytes can be carried out sequentially in time at one end of the long microchannel and gives information on the number of analytes present in the solution analyzed and their respective concentrations. This method is known for its very good resolution in separating two analytes, but has the drawback of diluting the analytes, which makes detection difficult or impossible in the case of analytes at a very low concentration.
To overcome this difficulty, a second method is known, called countercurrent gradient electrofocusing. This too employs a microfluidic network having at least two reservoirs connected to each other by channels and by at least one separation channel or chamber that concentrates the analytes in a separation channel when an appropriate pressure gradient and electric field are applied. To effect this preconcentration, there must be an electric field gradient in the central channel while the liquid flow rate is constant. Today there are several methods for creating a gradient (see Shackman and Ross, “Review Counterflow Gradient Electrofocusing,” Electrophoresis 2007, 28, 556-571).
FIG. 1A shows the value of the electric field as a function of the abscissa inside a separation channel and along its lengthwise axis, and FIG. 1B shows the value of the velocity of the charged particles as a function of the abscissa inside the same separation channel and along its lengthwise axis. This velocity is the sum of their electrophoretic velocity and the velocity of the liquid. As shown in these figures, with this method the velocity of each analyte varies within the network and may become zero at one spot in the network. Thus, each analyte contained in the network migrates to the spot where its velocity is zero, at which point it is preconcentrated. Two analytes with different electrophoretic mobilities can, in theory, thus be preconcentrated in two distinct locations in the microchannel. However, this method suffers from the fact that the preconcentration zones are too wide to achieve good separation of the analytes (the preconcentration zones overlap). Moreover, the stability of the ionic gradient is often difficult to obtain (thermal or electrokinetic instabilities). Thus, this technique is used more as an overall preconcentration tool before a separation. In this case we speak of electrocapture (see Shackman et al., FIG. 5) and today no technique exists for creating an electric field gradient that is fine and stable enough to ensure appropriate separation concomitantly with preconcentration.
Also known is US Patent Application 2006/018469 by Han et al. (hereinafter Han), which describes a method combining the advantages of the two above-described methods: it has a non-selective electrocapture step followed by an electrophoretic separation step. As shown in FIG. 2, this device has two U-shaped microchannels 1, 2 at the ends of each of which is a reservoir, respectively 4, 5, 6, and 7. The respective bases 8, 9 of these two microchannels are arranged in parallel and a nanochannel 10 connects the bases of the first and second microchannels 1, 2 at their median parts 11, 12.
Platinum electrodes 13, 14, 15, 16 are disposed in each of reservoirs 4, 5, 6, and 7 and are connected to at least one voltage generator, not shown, that generates a potential difference between them. Thus, a potential difference is generated between the inlet and the outlet of the nanochannel. With such a device, displacement of the analytes is effected electrically by electrophoresis and electro-osmosis.
Embodiments also provide the use of mechanical means able to generate a pressure difference between said reservoirs 4, 5, 6, and 7, the means may, for example, be comprised of one or more micropumps and ensure movement of the solution in the device. Embodiments also provide the alternating use of electrical means for moving the solution and mechanical means for moving the solution.
The first step associated with the method described in Han consists of creating a strong non-selective preconcentration of the sample by means of a space charge region used as a barrier. When a weak electric field parallel to nanochannel 10 is generated via the first generator, applying a voltage of 1 V to reservoirs 4 and 5 and a zero voltage to reservoirs 6 and 7, no preconcentration of the compound occurs. When the electric field is increased by increasing the voltage in reservoirs 4 and 5, displacement of the ions contained in the solution occurs to a limited degree and an ion-poor zone 17 forms in microchannel 1 at right angles to nanochannel 10 in which zone there are as many negative as positive ions. When a strong electric field is applied by turning up the voltage still higher in reservoirs 4 and 5, the neutrality of the sum of the ions present in said zone 17 is no longer preserved and a space charge region is created. If the voltage in reservoir 5 is then turned down so that it is equal to half that present in reservoir 4, a secondary electric field perpendicular to nanochannel 10 is generated. Displacement of the liquid contained in said first zone 17 then occurs by electro-osmosis. The charged analytes contained in the liquid are thus transported to the space charge region, which they are unable to penetrate. Accordingly these charged analytes build up in zone 18 of the microchannel located upstream of zone 17 and before the intersection between microchannel 1 and nanochannel 10.
After this non-selective preconcentration step, Han shows that it is possible to separate the preconcentrated analytes with different electrophoretic mobilities by capillary electrophoresis.
A device according to Han has a number of drawbacks. This device does not allow selective concentration of the analytes at the intersection between the microchannel and the nanochannel (global capture) and thus requires the use of another technique and an associated device, specifically capillary electrophoresis, to separate them. This is explained by the use of a nanochannel which creates an overly strong space charge zone. Also this device is found to have secondary electro-osmosis phenomena (see Han Physical Review Letters) that disturb the space charge zone responsible for preconcentration of the species contained in the solution. These phenomena are observed in particular when the space charge zone is created. They are also caused by the non-homogeneity of the electric field due to the orthogonal connection between the microchannel and the nanochannel.
The goal of the invention is to resolve these difficulties by proposing a method and a device for implementing this method enabling the preconcentration and separation steps to be coupled through a series of selective preconcentrations of the various charged analytes contained in an electrolyte.